Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.     

Reciprocal and non-reciprocal homologous recombination between Escherichia coli chromosomal DNA and ultraviolet light-irradiated plasmid DNA

Plasmid DNA substrates were used to study ultraviolet (UV)-induced recombination events in Escherichia coli host cells. Plasmids derived from pBR322, containing all or part of the lac operon of E. coli, were irradiated with ultraviolet light before transformation into E. coli strains of different recA and lacY genotypes. Recombinational exchanges were identified by phenotypic changes in lactose utilization and were confirmed by restriction analysis of isolated plasmids. Ultraviolet-induced reciprocal plasmid-chromosome recombination occurred at a slightly higher frequency then non-reciprocal chromosome-to-plasmid recombination, and at a much higher frequency than non-reciprocal plasmid-to-chromosome recombination. These frequencies did not depend on segregative mechanisms. The asymmetry of non-reciprocal exchange was not due to the particular arrangement of wild-type and lacY1 alleles because the same results were observed when these were interchanged. The host recA gene was required for plasmid-chromosome recombination, and slightly enhanced plasmid survival. Evidence for plasmid replication prior to recombination was found in reciprocal recombinants, but rarely in the non-reciprocal recombinants analyzed. Irradiation of competent bacterial host cells prior to transformation did not effectively induce plasmid-chromosome recombination.     

Homologous recombination in a mammalian plasmid

Summary Bovine papillomavirus (BPV) shuttle vectors replicate as a circular plasmid in mouse cell nuclei without impairing host cell viability. We used these vectors to analyze homologous recombination in mammalian cells. When several BPV-based plasmids carrying direct repeats were introduced into C127 cells, we detected many recombinant plasmid molecules that have lost the sequence between the repeats. Many recombinant type molecules as well as parental type molecules were detected in all the cell clones isolated for analysis. Sequencing after rescue of the plasmid inEscherichia coli showed that most of the recombinants were from accurate homologous recombination. When the repeats on the plasmid were in inverted orientation, no crossing-over type products were detected. We discuss possible mechanisms that explain these features.     

Homologous Recombination between Autonomously Replicating Plasmids in Mammalian Cells

The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neoR colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.     

Homologous recombination and mutagenesis of gamma-irradiated plasmid DNA in Escherichia coli host cells

Plasmid DNA was used to study gamma-radiation-induced recombination and mutagenesis in Escherichia coli host cells. Plasmid pBRP1, a derivative of pBR322 containing the lac operon of E. coli, was irradiated with 60Co gamma rays prior to transformation into E. coli strains of different recA and lac genotypes. Plasmid-chromosome recombination was assayed in lacY1 host cells, whereas plasmid mutagenesis was assayed in delta lac host cells lacking chromosomal sequences homologous to the plasmid. Both recombinant and mutant plasmids were identified by the phenotypic changes in lactose utilization, and confirmed by restriction analysis of isolated plasmids. Plasmid-chromosome recombination was induced to high levels (about 20% of survivors at 700 Gy) and was dependent on the host recA gene. Plasmid mutagenesis occurred at lower levels (about 1.5% of survivors at 600 Gy) and was relatively independent of the recA gene. Plasmid survival was unaffected by the presence or absence of host recA mutations or the potential for plasmid-chromosome recombination.     

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.     

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.     

Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo.

Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo. In this method 'probe' sequences are inserted in a very small plasmid vector and introduced into recombination-proficient bacterial cells. Genomic bacteriophage libraries are propagated on the cells, and phage bearing sequences homologous to the probe acquire an integrated copy of the plasmid by reciprocal recombination. Phage bearing integrated plasmids can be purified from the larger pool of phage lacking plasmid integrates by growth under the appropriate selective conditions.     

利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法     

Transformation depending on intermolecular homologous recombination is stimulated by UV damage in transfected DNA

DNA-mediated gene transfer (DMGT) was performed in DNA repair-proficient and UV-hypersensitive, repair-deficient Chinese hamster ovary (CHO) cell lines using the UV-irradiated thymidine kinase gene from herpes simplex virus (HSV-TK). Transformation frequencies in repair-deficient CHO cell lines declined relative to repair-proficient cells with increasing UV damage in transfected DNA; approximately 3-fold higher UV fluence was required to inactivate 50% of irradiated HSV-TK plasmid molecules in repair-proficient cells. In cotransfection experiments performed with pairs of HSV-TK plasmids containing linker insertion mutations in TK coding sequences, moderate UV damage in plasmid DNA enhanced the yield of TK+ transformants resulting from homologous recombination between HSV-TK sequences up to 4-fold. These results suggest that UV damage in DNA can stimulate transformation of mammalian cells dependent on intermolecular DNA homology.     

Double-strand gap repair in a mammalian gene targeting reaction.

To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments.     

携带PTEN基因的重组腺病毒表达载体构建的研究

构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础.     

Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells.

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.     

细菌内同源重组法制备FMDV聚蛋白编码基因重组腺病毒

采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.     

人核糖核酸酶抑制因子基因重组腺病毒载体的构建及鉴定

目的构建含有人核糖核酸酶抑制因子（hRI）基因的重组腺病毒载体。方法以含有全长cDNA的pT7-RI为模板,PCR扩增hRI,经T载体克隆后,酶切亚克隆到穿梭质粒pAdTrack—CMV上,在BJ5183细菌内和pAdEasy-1同源重组。筛选阳性克隆,酶切、PCR及测序鉴定,线性化后脂质体法转染293细胞进行包装、扩增。通过观察绿色荧光蛋白（GFP）的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。结果酶切鉴定及PCR结果证明hRI基因重组腺病毒载体构建成功,病毒滴度为1．5×10^10 pfu/ml。结论应用细菌内同源重组法成功构建了含hRI基因的重组腺病毒载体。